Assay for transmissible spongiform encephalopathies

ABSTRACT

Provided is a novel method for detecting Transmissible Spongiform encephalopathies (TSE). The method comprises: selecting a sera sample from a subject to determine whether the subject has transmissible spongiform encephalopathy; and detecting antibodies that immune react with  Spiroplasma  proteins in the sample. The method detects antibodies in the individual sera samples that react with recombinant  Spiroplasma -specific Hsp60 by standard ELISA methodology. This method provides for rapid detection of TSE by determination of presence of antibodies in test sera showing associated  Spiroplasma  infection that has become consistent with presence of TSE. The method provides a mechanism to detect presence of TSE without invasive techniques currently needed to make diagnosis using brain tissues for prion detection. This method provides means of detection of TSE in the live patient without need for using necropsy tissues. This method provides the first method discovered that detects antibodies against TSE infection-specific proteins in sera or cerebrospinal fluid samples.

This application claims priority from U.S. Provisional Application Ser. No. 60/573,814 (“the '814 application”) filed May 24, 2004. The '814 application is incorporated herein by reference.

This work was supported by NIH NINDS 5RO1NS044000-O3 to Frank O. Bastian.

BACKGROUND OF THE INVENTION

The present invention presents an assay for transmissible spongiform encephalopathies in humans and other animals.

Transmissible spongiform encephalopathies (“TSE”) are diseases that can affect humans and other animals. It is characterized by spongy degeneration of the brain. The disease is known as scrapie in sheep and goats. The condition is known as both bovine spongiform encephalopathy or mad cow disease in cattle. There exist human variants of the disease known as Kuru, Creutzfeldt-Jakob Disease (“CJD”), fatal familial insomnia, and Gerstmann-Strausler disease. Deer and elk are known to contract a variant of the disease known as chronic wasting disease. TSE is seen in farmed mink, known as transmissible mink encephalopathy.

At a molecular level, the disease is characterized by deposition of prion protein in brains from TSE-afflicted humans or animals. The prion protein is a normal constituent of brain tissue. In individuals affected by transmissible spongiform encephalopathies, there is conformational change in the protein making it resistant to denaturation by proteolytic enzymes. Since the prion proteins of affected individuals are resistant to most methods of denaturation, most assays for the diseased version of the protein seek to differentiate between the diseased and normal version of the protein. Typical assays for the protein first treat suspected brain tissue with proteolytic enzymes, then seek to identify the prion proteins (usually by polyacrylamide gel electrophoresis followed by western blotting with an antibody specific for both types of prion proteins). In unaffected individuals, there is often no protein available for recognition by the antibodies during western blotting following proteolytic digestion. Because of the ability of diseased prion proteins to resist proteolytic denaturation, they are recognized by the anti-prion antibodies. An alternate approach is to use antibody recognition of the prion protein by Enzyme-Linked ImmunoadSorbent Assay (“ELISA”) using antibodies tagged with enzymes or fluorescent molecules. In either case, a fluorescent or calorimetric signal can be used to conclude the testing. Abnormal Prion has not been detected in sera or cerebrospinal fluid derived from TSE-afflicted individuals.

Current methods of testing for the presence of transmissible spongiform encephalopathies are for the most part conducted post-mortem. This is because an analysis of the prion proteins of the individuals must be done and this is accomplished, by an analysis of brain tissue or other tissues such as tonsil. Thus, such analyses are extremely invasive negating applicability to preclinical diagnosis of TSE. These assays, while useful, suffer from their reliance on brain tissue to provide a diagnostic result. The use of brain material is not feasible for screening. This is especially not suitable when the individual to be tested is a living human or for the purpose only to determine if the individual is a safe blood donor. Therefore there exists a need to develop a test that can provide diagnosis in a living individual with minimal invasion, preferably using sera samples.

An alternate approach has led to a better understanding of the pathogenesis of TSE whereby a wall-less bacterium called Spiroplasma is closely associated with these diseases. The presence of Spiroplasma was initially discovered in an ultrastructural study of a brain biopsy obtained from a 46 year old CJD patient. Since then, the presence of Spiroplasma genes in TSE brain tissues has been shown using molecular techniques including polymerase chain reaction (PCR), Southern blotting and DNA sequence analyses. More recently, a unique Spiroplasma species from TSE-infected brain tissues has been isolated by passage through embryonated eggs into cell-free media. The role of Spiroplasma infection in the pathogenesis of TSE is supported by recent studies that have shown the normal prion isoform to serve as a receptor protein for a bacterium. It is presumed that interaction of Spiroplasma with the prion results in the disease and accumulation of the misfolded prion protein.

The occurrence of a consistent Spiroplasma infection in association with individual TSE cases provided the opportunity to develop a serum test for the disease based upon the presence of antibodies generated against Spiroplasma proteins. Heat shock protein 60 (Hsp60) was chosen because of prior data that showed interaction of bacterial-specific Hsp60 protein on the bacterial surface with the prion receptor. It is noteworthy that Hsp60 is widespread in nature. However, the Hsp60 of bacteria is specific in that there is 70% homology with other bacteria, but a 100% homology among strains of the same genus. There is 50% homology of bacterial Hsp60 with human Hsp60 proteins. The inventor has isolated the Hsp60 gene specific for Spiroplasma, produced recombinant Spiroplasma-specific Hsp60 recombinant protein and shown reactivity with sera from individual TSE cases by using ELISA.

The current methods of testing are also inefficient when applied to large numbers of livestock. Brain or neural material must be taken after slaughter and processed to be assayed. This delay can result in the carcass of the animal being placed into the human or animal food supply before testing can be concluded. Also, these postmortem assays cannot be used to test and produce groups of animals that are free of transmissible spongiform encephalopathies or insure that animals with transmissible spongiform encephalopathy do not co-mingle with disease free animals. The use of brain tissue, in addition to mandating a postmortem test, is also very inconvenient. An assay using a more readily available bodily fluid such as sera or tissue offers a more convenient approach to testing of any animal type for TSE.

SUMMARY OF THE INVENTION

The present invention relates to methods of detecting transmissible spongiform encephalopathies in animals, including human beings. The invention relies on the use of a protein of the Spiroplasma bacterium to identify individuals affected with transmissible spongiform encephalopathies. Specifically, this invention uses the heat shock protein 60 (Hsp60) of Spiroplasma mirum. Alternatively, other proteins of Spiroplasma mirum could be used in place of Hsp60 in this assay. Additionally, the proteins of other members of the genus Spiroplasma could be used to create the assay. Artificially synthesized peptides representing amino acid fragments of proteins from the genus Spiroplasma may also be used.

The present invention uses the serum of an animal or human being as its testing material. In another embodiment of the invention, whole blood may be used as the testing material. In yet another embodiment of the present invention, cerebrospinal fluid of an animal or human being may serve as the testing material. Other body fluids such as urine, tears and saliva may also serve as a testing material.

It is an object of the invention to provide a method of detecting a transmissible spongiform encephalopathy (TSE) disease in a mammal, including a human, a cow and a sheep, comprising detecting the presence of an antibody to a protein from a bacterium of the genus Spiroplasma in the serum of the mammal.

It is an object of this invention to provide a method of detecting a transmissible spongiform encephalopathy (TSE) disease in a mammal, including a human, a cow and a sheep, comprising contacting a serum sample from a mammal with at least a portion of heat shock protein 60 (Hsp60) from a Spiroplasma bacterium and detecting a reaction product of Hsp60 or a portion thereof with a component of the plasma.

It is a further object of this invention to provide a method of diagnosing a TSE disease in a mammal comprising detecting the presence of a Spiroplasma infection in a mammal.

It is a further object of this invention to provide a method of diagnosing a TSE disease in a mammal comprising detecting an antibody to Spiroplasma Hsp60 in a serum sample from a mammal.

It is a further object of this invention to provide a method of diagnosing a TSE disease in a mammal comprising detecting an antibody to Spiroplasma Hsp60 in a cerebrospinal fluid sample from a mammal.

It is a further object of this invention to provide a method of diagnosing a TSE disease in a mammal comprising detecting an antibody to Spiroplasma Hsp60 in a whole blood sample from a mammal.

It is a further object of this invention to provide an isolated Hsp60 protein and an isolated polynucleotide encoding an Hsp60 protein having DNA and amino acid sequences of SEQ ID NO. 1, and SEQ ID NO. 2, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustrative gel electrophoresis of polymerase chain reaction (PCR) generated amplified products obtained by probing TSE and normal brain tissues using oligonucleotide primers specific for Spiroplasma Hsp60 gene. The PCR amplified Spiroplasma Hsp60 gene product is only present in the TSE infected brain but not in normal brain or water control.

FIG. 2 is an illustrative acrylamide electrophoresis of purified recombinant Spiroplasma Hsp60 protein. The 45 kDa GST-tagged recombinant Spiroplasma Hsp60 protein eluted from the glutathione column is soluble and in sufficiently pure form (lane 2) for development of an indirect ELISA.

FIG. 3 is a statistical analysis variance table (absorbance at 490 nm of the indirect ELISA plates) showing significant evidence of circulating antibodies against Spiroplasma recombinant Hsp60 protein (p-value<0.0001) in sera from CJD patients.

FIG. 4 is a statistical analysis variance table (absorbance at 490 nm of the indirect ELISA plates) showing significant evidence of circulating antibodies against Spiroplasma recombinant Hsp60 protein (p-value<0.0001) in sera from scrapie-infected sheep.

DETAILED DESCRIPTION OF THE INVENTION

The gene for heat shock protein 60 (Hsp60) of Spiroplasma mirum (GenBank ID M24662) was identified by polymerase chain reaction (PCR) and DNA sequence analysis. The gene was cloned in a Topo vector (Invitrogen). The DNA and predicted amino acid sequence of Spiroplasma mirum Hsp60 is not in the GenBank, has not been published, and is claimed here as part of this invention as described in SEQ ID NO 1 and SEQ ID NO 2. New oligonucleotide primers were designed from this novel sequence and used to probe DNA extracts from TSE brain tissues. As depicted in FIG. 1, which has a series of lanes 10 marked 1 through 7, two Spiroplasma-specific Hsp60 probes revealed presence of amplified gene products in TSE brain tissues but not in normal brain tissues or a water control. FIG. 1 shows presence of 130 and 140 bp amplified PCR products in TSE-infected brains and not in controls. Of the lanes 10, the lane marked 1 shows 100 bp markers. The lanes marked 2 and 5 show water controls. The lanes marked 3 and 6 show normal brain and the lanes marked 4 and 7 show TSE-infected brain. The amplified PCR products are the contrasting bands in the lanes marked 4 and 7. Other non-specific banding represents primer/dimer.

The nucleic acid sequence encoding Spiroplasma mirum Hsp60 was placed into a suitable bacterial expression system using GST fusion technology (Novagen). Lysates of bacteria expressing GST bound Hsp60 were used to isolate recombinant Hsp60 of Spiroplasma mirum purified by passing through a glutathione column. The recombinant Hsp60 was eluted from the column as a soluble fraction. FIG. 2, which has lanes 11 marked 1, 2 and 3, shows purified soluble fractions of recombinant Hsp60 of 45 kDa size. FIG. 2 shows elution fractions of GST-bound Spiroplasma Hsp60 in lanes 11 marked 2 and 3 following passage over a glutathione column. The lane marked 1 shows markers, and the lanes marked 2 and 3, show soluble 45 kDa recombinant Spiroplasma Hsp60 protein.

Purified Spiroplasma mirum recombinant Hsp60 was attached to the surface of microwell plates. The wells of the plates were blocked with a commercially available blocking buffer. Serum samples from individual patients who were Creutzfeldt-Jakob Disease positive or controls were added to the wells containing the Hsp60 protein. Following an incubation period, the serum samples were removed and each well was washed three times with either tris buffered saline or phosphate buffered saline. A goat anti-human antibody tagged with horseradish peroxidase was added to the wells. Following an incubation period, the wells were washed. A calorimetric substrate was added to the wells. In the presence of horseradish peroxidase, the substrate is oxidized to produce a colored substrate. The amount of analyte produced can be determined by measuring the absorbance of individual samples or wells at the wavelengths produced by the oxidized substrates. All studies were done in triplicate along with three wells with no primary antibody for determining background. An alternate approach would be to use a chemiluminescent substrate of horseradish peroxidase. In such a case, the amount of an analyte can be measured by the relative light units produced.

Statistical analysis variance tables (absorbance at 490 nm of the indirect ELISA plates) showed significant evidence of circulating antibodies against Spiroplasma recombinant Hsp60 protein (p-value<0.0001) in sera from CJD patients. FIG. 3 compares absorbance readings at 490 nm of thirty individual CJD sera samples compared to thirty individual normal sera. Column 12 shows distribution of plate readings at 490 nm of thirty individual CJD sera showing mean 15 and standard error range 16. Column 13 shows distribution of plate readings at 490 nm of thirty individual normal human sera showing mean 17 and standard error range 18. Column 14 shows plate readings 19 at 490 nm of plate background (no primary antibody).

Statistical analysis variance tables (absorbance at 490 nm of the indirect ELISA plates) showed significant evidence of circulating antibodies against Spiroplasma recombinant Hsp60 protein (p-value<0.0001) in sera from scrapie-infected sheep. FIG. 4 compares absorbance readings at 490 nm of ten individual scrapie sheep sera samples compared to forty individual normal sheep sera. Column 20 shows distribution of plate readings at 490 nm of ten individual scrapie sera showing mean 23 and standard error range 24. Column 21 shows distribution of plate readings at 490 nm of forty individual normal sheep sera showing mean 25 and standard error range 26. Column 22 shows plate readings 27 at 490 nm of plate background (no primary antibody).

The statistical analyses of the ELISA results from the study of thirty individual CJD sera show that there is a >95% confidence in identifying an individual CJD case and a >95% confidence in identifying an individual normal control. Similarly, the study of individual scrapie sera show a >95% confidence in identifying an individual scrapie-infected animal and a >95% confidence in identifying an individual normal control animal. 

1. A method of detecting a transmissible spongiform encephalopathy (TSE) disease in a mammal comprising detecting the presence of an antibody to a protein from a bacterium of the genus Spiroplasma in the serum of the mammal.
 2. A method of detecting a transmissible spongiform encephalopathy (TSE) disease in a mammal comprising contacting a serum sample from a mammal with at least a portion of heat shock protein 60 (Hsp60) from a Spiroplasma bacterium and detecting a reaction product of Hsp60 or a portion thereof with a component of the plasma.
 3. A method of diagnosing a TSE disease in a mammal comprising detecting the presence of a Spiroplasma infection in a mammal.
 4. A method of diagnosing a TSE disease in a mammal comprising detecting an antibody to Spiroplasma Hsp60 in a serum sample from a mammal.
 5. A method of diagnosing a TSE disease in a mammal comprising the sequential steps of (a) binding at least a portion of a Spiroplasma Hsp60 protein to a solid substrate to form a diagnostic substrate; (b) contacting the diagnostic substrate with serum from a mammal to bind to the diagnostic substrate a primary antibody specific for Hsp60 present in the serum; (c) washing the diagnostic substrate to substantially remove proteins from the plasma which are not bound to the diagnostic substrate; (d) contacting the diagnostic substrate with a secondary antibody specific for the primary antibody of the mammal but not specific for Hsp60 protein to bind the secondary antibody to at least a portion of any primary antibody bound to the diagnostic substrate; (e) washing, the diagnostic substrate to substantially remove non-bound secondary antibody; and (f) measuring the amount of secondary antibody bound to the diagnostic substrate relative to a standard.
 6. A method according to claim 1 wherein the mammal is a human.
 7. A method according to claim 2 wherein the mammal is a human.
 8. A method according to claim 3 wherein the mammal is a human.
 9. A method according to claim 4 wherein the mammal is a human.
 10. A method according to claim 5 wherein the mammal is a human.
 11. A method according to claim 1 wherein the mammal is a cow.
 12. A method according to claim 2 wherein the mammal is a cow.
 13. A method according to claim 3 wherein the mammal is a cow.
 14. A method according to claim 4 wherein the mammal is a cow.
 15. A method according to claim 5 wherein the mammal is a cow.
 16. A method according to claim 1 wherein the mammal is a sheep.
 17. A method according to claim 2 wherein the mammal is a sheep.
 18. A method according to claim 3 wherein the mammal is a sheep.
 19. A method according to claim 4 wherein the mammal is a sheep.
 20. A method according to claim 5 wherein the mammal is a sheep.
 21. A method of detecting a transmissible spongiform encephalopathy (TSE) disease in a mammal comprising detecting the presence of an antibody to a protein from a bacterium of the genus Spiroplasma in the cerebrospinal fluid of the mammal.
 22. A method of detecting a transmissible spongiform encephalopathy (TSE) disease in a mammal comprising contacting a cerebrospinal fluid sample from a mammal with at least a portion of heat shock protein 60 (Hsp60) from a Spiroplasma bacterium and detecting a reaction product of Hsp60 or a portion thereof with a component of the sample.
 23. A method of diagnosing a TSE disease in a mammal comprising detecting an antibody to Spiroplasma Hsp60 in a cerebrospinal fluid sample from a mammal.
 24. A method of diagnosing a TSE disease in a mammal comprising the sequential steps of (a) binding at least a portion of a Spiroplasma Hsp60 protein to a solid substrate to form a diagnostic substrate; (b) contacting the diagnostic substrate with cerebrospinal fluid from a mammal to bind to the diagnostic substrate a primary antibody specific for Hsp60 present in the cerebrospinal fluid; (c) washing the diagnostic substrate to substantially remove proteins from the cerebrospinal fluid which are not bound to the diagnostic substrate; (d) contacting the diagnostic substrate with a secondary antibody specific for the primary antibody of the mammal but not specific for Hhsp60 protein to bind the secondary antibody to at least a portion of any primary antibody bound to the diagnostic substrate; (e) washing, the diagnostic substrate to substantially remove non-bound secondary antibody; and (f) measuring the amount of secondary antibody bound to the diagnostic substrate relative to a standard.
 25. A method according to claim 21 wherein the mammal is a human.
 26. A method according to claim 22 wherein the mammal is a human.
 27. A method according to claim 23 wherein the mammal is a human.
 28. A method according to claim 24 wherein the mammal is a human.
 29. A method according to claim 21 wherein the mammal is a cow.
 30. A method according to claim 22 wherein the mammal is a cow.
 31. A method according to claim 23 wherein the mammal is a cow.
 32. A method according to claim 24 wherein the mammal is a cow.
 33. A method according to claim 21 wherein the mammal is a sheep.
 34. A method according to claim 22 wherein the mammal is a sheep.
 35. A method according to claim 23 wherein the mammal is a sheep.
 36. A method according to claim 24 wherein the mammal is a sheep.
 37. An isolated Hsp60 protein having the amino acid residue sequence of SEQ ID NO:
 2. 38. An isolated polynucleotide encoding an Hsp60 protein having the amino acid residue sequence of SEQ ID NO:
 2. 39. An isolated polynucleotide having the nucleotide sequence of SEQ ID NO:
 1. 